PURPOSE: Fanconi anemia complementation group F (FANCF) is a key factor to maintaining the function of Fanconi anaemia/BRCA (FA/BRCA) pathway, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In the present study, we evaluated the chemosensitization effect of FANCF in breast cancer cells. METHODS: We performed specific knockdown of the endogenous FANCF in breast cancer cells by transfecting the cells with an FANCF short hairpin RNA (shRNA) vector. Cell viability was measured with a Cell Counting Kit-8, and DNA damage was assessed with the alkaline comet assay. The apoptosis, cell cycle, and drug accumulation were measured by flow cytometric analysis. Protein expression levels were determined by Western blot analysis, using specific antibodies. RESULTS: The analyses of two breast cancer cell lines (MCF-7 and MDA-MB-435S) demonstrated that the FANCF shRNA could effectively block the FA/BRCA pathway through the inhibition of Fanconi anemia complementation group D2 ubiquitination. Moreover, FANCF silencing potentiated the sensitivity of cells to mitomycin C (MMC), where combined FANCF shRNA/MMC treatment inhibited cell proliferation, induced S-phase arrest, apoptosis, and DNA fragmentation, and reduced the mitochondrial membrane potential, compared with MMC treatment alone. CONCLUSION: Taken together, this study demonstrates that the inhibition of FANCF by its shRNA leads to a synergistic enhancement of MMC cytotoxicity in breast cancer cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of breast cancer.
Apoptosis ; Blotting, Western ; Breast ; Breast Neoplasms ; Cell Count ; Cell Cycle ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Comet Assay ; Complement System Proteins ; DNA Damage ; DNA Fragmentation ; Fanconi Anemia ; Fanconi Anemia Complementation Group F Protein ; Membrane Potential, Mitochondrial ; Mitomycin ; RNA, Small Interfering ; Ubiquitin ; Ubiquitination

OBJECTIVE To study whether the ciprofloxacin(CIP)combined with amikacin(AMK)will decrease the drug-resistant mutants of Escherichia coli(ECO)in vitro.METHODS The MIC of CIP and AMK alone was determined by agar plates dilution method,and the combined MIC by checkerboard method.The mutant prevention concentration(MPC)both alone and combined was determined by the method of agar plates dilution method,and then the value of selectiveity index(SI)(MPC/MIC)would be acquired.RESULTS The SI of two antibiotics separatedly were 16(CIP)and 32(AMK).When two antibiotics combined,if the concentration of AMK and CIP reached 2MIC could restrain the mutants.After these two antibiotics combined,the value of SI(MPCcombined/MICcombined)came to be 8(0.008/0.001)and 8(2.0/0.25).CONCLUSIONS Compared with alone,CIP and AMK combined can decrease the MPC and SI to ECO,and have the more effect to AMK.In this way,we can reduce the drug-resistant mutants.

BACKGROUNDSerine threonine kinase 15 (STK15) is a kind of mitotic kinase. The overexpression of STK15 is significantly associated with carcinogenesis in many tumors, however, its expression and significance in human lung cancer are still unclear. The aim of this study is to investigate the expression of STK15 in squamous cell carcinoma and adenocarcinoma of the lung and to analyze the correlation between STK15 expression and clinicopathological factors.

METHODSThe pattern of STK15 protein expression was detected in 44 squamous cell carcinomas, 36 adenocarcinomas and 20 paracancerous lung tissue samples by immunohistochemistry method using anti-STK15 antibody. The relative quantity of STK15 protein expression was detected by Western blot, and STK15 mRNA expression was detected by RT-PCR in 40 fresh lung cancer samples and corresponding paracancerous lung tissues.

RESULTSPositive expression rate of STK15 protein was 68.75% (55/80) in lung cancer tissues and 0% in paracancerous controls (P < 0.001). STK15 expression was significantly related to differentiation grade of lung cancer (P=0.011), but not to histological classification, TNM stages or lymphatic metastasis (P > 0.05). The relative expression levels of STK15 protein (P < 0.001 ) and STK15 mRNA (P < 0.001) in lung cancer tissues were both significantly higher than those of corresponding paracancerous lung tissues.

CONCLUSIONSThe expression of STK15 protein and STK15 mRNA is significantly higher in lung cancer tissues than that in paracancerous lung tissues. The expression of STK15 correlates with differentiation of lung cancer.

BACKGROUNDThere are lots of mucus, blood, inflammatory cells and necrotic material in the pick-and-smear slides, resulting in a low detection rate. Liquid-based cytologic test (LCT) has been applied for cervical cytology diagnosis successfully and widely, however it is few reported yet for sputum cytology diagnosis at present. The aim of this study is to evaluate the clinical value of LCT in sputum examination of patients with lung cancer, and to find a novel method of early diagnosis of lung cancer.

METHODSThe cytologic findings and the diagnostic rate for lung cancer were compared between LCT and conventional pick-and-smear method.

RESULTSThere were smaller area of smear membrane, clearer background, more distinctly cytologic picture and stereoscopic fell by LCT comparing with pick-and-smear method. The diagnostic rate for small cell lung cancer by LCT was significantly higher than that by pick-and-smear method (P < 0.05). After combined detection of the two methods, the diagnostic rate for lung cancer was obviously improved (85.1%), which was remarkably higher than that by pick-and-smear method alone (P < 0.01).

CONCLUSIONSIt is operated easily for LCT to be well controlled in making smear and dyeing. LCT may be a novel technique worthy of wide use. Combination of LCT with pick-and-smear method appears to be of great value in clinical application.

BACKGROUNDIntegrin-linked kinase (ILK) is a Ser/Thr protein kinase. Many studies have showed that ILK was closely related to occurrence, proliferation, invasion and metastasis in many malignant tumors, and it appeared to be an upstream cross point of tumor-associated factors. The aim of this study is to explore the relationship between the expression of ILK and some clinical pathological factors in human non-small cell lung cancer (NSCLC), and analyze whether there is relativity between ILK and E-cadherin.

METHODSImmunohistochemical S-P method was adopted to detect the expression of ILK and E-cadherin proteins in 76 NSCLC cases with the neighboring noncancerous tissue, and the expressions of them in 30 fresh NSCLC samples were determined with Western Blot assay.

RESULTSImmunohistochemically, the overexpression of ILK protein in NSCLC was 53/76 (69.7%), including 33/44 (75.0%) squamous cell carcinoma and 20/32 (62.5%) adenocarcinoma, but its expression was not related to the histological type (P= 0.247 ). Expression of ILK was related to differentiation (rs=-0.296, P=0.009), lymph node metastasis (rs=0.311, P=0.006) and clinical stage (rs=0.350, P=0.002). Moreover, Kaplan-Meier survival estimates showed a significant correlation between ILK expression and patient survival in Log-rank test (P=0.006). Overexpression of ILK in NSCLC was associated with unfavorable prognosis. An inverse correlation between the levels of ILK and E-cadherin was found (rs=-0.514, P < 0.001). Western Blot result showed that the level of ILK in the tumor tissues was noticeably higher than that in the normal tissues (t=-6.811, P=0.0002), and an inverse correlation between the levels of ILK and E-cadherin was proved (P=0.001).

CONCLUSIONSIn NSCLC, ILK can interact with some tumor-associated factors, through which it appears to be involved in several oncogenesis-related events ,including promotion of cell survival ,as well as cell migration and invasion.ILK keeps significant inverse correlation to E-cadherin,andit would be one of the pathways for ILK to affect differentia-tion,clinical stage ,lymph node metastasis and prognosis of patients .ILK expression can be a useful predictorof poor prognosis in NSCLC,and the detections of ILK and E-cadherin will help us better to predict prognosisof patients .

BACKGROUNDTo study the relationship between human thymosin β4 (Tβ4 )expression and biological factors regarding invasion, metastasis and prognosis of human non-small cell lung cancer (NSCLC).

METHODSTβ4 expression was detected in samples of 76 paraffin-embedded specimens with the neighboring noncancerous tissue using anti-Tβ4 IgY antibody (primary antibody) by immunohistochemical staining. The relationship between Tβ4 expression and the clinicopthological factors was analyzed by Chi-square test, multivariate analysis and Kaplan-Meier method.

RESULTSIn immunoreactive cells, staining was mainly located in cytoplasma. Human Tβ4 expression was high positive in lung cancer tissues (76.3%, 58/76) while low positive in normal lung tissues. Tβ4 expression was positively associated with TNM stage (r=0.239, P=0.032), lymphatic metastasis (r=0.243, P=0.029) and venous metastasis (r=0.224, P=0.045).A negative correlation was found between Tβ4 expression and cell differentiation (r=-0.368, P=0.002). Patients with high Tβ4 expression had a worse prognosis than those with low Tβ4 expression (P < 0.05)

CONCLUSIONSLung cancer has overexpression of Tβ4, which is closely related to TNM stages, cell differentiation, metastasis of the cancer and prognosis of the patients with lung cancer. Detection of Tβ4 expression in lung cancer tissues might be helpful to predict prognosis of patients with lung cancer.

BACKGROUNDTo study the relationship between human heparanase expression and biological factors regarding invasion, metastasis and prognosis of human non-small cell lung cancer (NSCLC).

METHODSThe expression of heparanase was assessed in 122 paraffin-embedded specimens and 38 freshly-taken tissues by immunohistochemical staining and Western blot. The relationship between heparanase expression and the clinicopathological factors was analyzed by Chi square test, multivariate analysis and Kaplan-Meier method.

RESULTSIn the immunoreactive cells, staining was mainly located in cytoplasma and membrane. Human heparanase was highly expressed in lung cancer tissues (78.7%, 96/122) while negative in epithelia of normal lung tissues. The level of heparanase was remarkably higher in NSCLC than that in normal tissues ( P = 0.043 ). Expression of heparanase significantly correlated with TNM stage ( P =0.025), lymphatic metastasis ( P =0.002) and vascular invasion ( P =0.000 3). The patients with positive heparanase expression had a significantly shorter survival than those with negative heparanase expression ( P =0.000 6). In multivariate analysis, only p-TNM stage, lymphatic metastasis and vascular invasion could be considered as prognostic factors.

CONCLUSIONSHeparanase might play an important role in the development, invasion and metastasis of NSCLC. It is indicated that patients with positive heparanase expression would have a greater chance of metastasis and a poorer prognosis. However, heparanase expression is not an independent prognostic factor.

Objective In order to explore the possibility of autologous transplantation of olfactory mucosa for spinal cord repair,the changes of autologous olfactory mucosa being transplanted into spinal cord and the effect of promoting axon regeneration in adult rats were investigated. Methods Forty male adult rats were divided into two groups randomly:olfactory mucosa transplantation and control groups.Olfactory mucosa (1*!mm+2) was taken from the posterior region of nasal septum,and this graft was transplanted into the posterior funiculus of cervical 1 segment of spinal cord and destructed the corticospinal tract(2*!mm?1^5*!mm).The control graft was derived from the respiratory lamina and then was grafted into the corresponding lesion.After being survived for 4-6 weeks,the animals were killed,the transplanted site was sectioned horizontally,immunofluorescence double labeled with the neurofilament(NF) and anti-NGFR-p75,investigated under the confocal microscope.Another set of slice was used immunohistochemistry to investigate the olfactory mucosa how to integrate with the surrounding tissue. Results The control lesions have the obvious hollow,no p75-positive reaction in the respiratory lamina and no NF-positive fibers grew through it;olfactory mucosa group showed that olfactory mucosa which is labeled p75-positive healed up with surrounded tissue,the hollow declined markedly,the labeled olfactory ensheathing cells(OECs) soakage into the nerve tissue.OECs induced a fine,unbranched,elongative growth of the cut CST axons.The axons were regenerated through the graft and continued to reenter into the caudal host part.Conclusion Autologous olfactory mucosa may be the donor which could repair the spinal cord injury for clinical situations.

Objective On the basis that olfactory ensheathing cells (OECs) transplanted into injured spinal cord may facilitate axonal regeneration, the OECs were cultured from olfactory bulb and nasal olfactory mucosa in the present study, in order to explore if the olfactory mucosa could be a new donation for transplanting the olfactory ensheathing cells. Methods OECs were harvested from olfactory bulb and mucosa based on the differing rates of attachment of the various cell types, following GFAP and NGFRp75 immunocytochemistry. Results Three morphological and immunohistochmically distinct types of cell which appeared bipolar,tripolar and flat morphology were present in primary cultures of adult rat olfactory bulb and olfactory mucosa.Conclusion The method of purification for OECs based on the different rates of attachment among the various cell types is simple, inexpensive and practical. The OECs from nasal olfactory mucosa like ones from the olfactory bulb is an accessible source of tissue for autologous grafting in human spinal paralegia in the future.

Objective To investigate if the cell death in hippocampus can occur after single and kindled audiogenic seizure,and the pertinent mechanisms involved. Methods HE staining was used to show the distribution of the dead cells and the immunocytochemistry was used to show the expression of the apoptosis\|related proteins Bcl\|2 and Bax. Results After single audiogenic seizure,there were a few dead cells in hippocampus.After kindled seizure a lots of pyknotic,acidophilic cells were found in hippocampal CA\-1,CA\-2,CA\-4 subregions and the granular layer of dentate gyrus.The CA (corrected optic density) values of Bax immunoreactivities in CA\-2 and CA\-4 subregions were increased significantly in kindled rats, in contrast with that in control rats.Conclusions\ Audiogenic kindling can induce marked cell death in hippocampus.Apoptosis may be one of the mechanisms underling this kind of cell death.